The mechanism of the phosphorylation-dependent myosin-linked regulation of smooth muscle myosin is being investigated. One aspect of this regulation involves understanding how myosin light chain kinase and phosphatases interact with myosin or actin. To do this we have measured the binding constants of the kinase and phosphatases to actin and to myosin in the hope of identifying their possible intracellular locations. We have also developed a method for determing light chain binding sites on myosin or proteolytic subfragments of myosin by incubating labelled light chains with polyacrylamide gels containing myosin (or its subfragments) which have been fixed and extensively washed to remove sodium-dodecyl-sulfate. This technique shows that both types of light chains bind to a region of the myosin head located about 76,000-100,000 Da in sequence from the N-terminal end. A third project utilizes a novel motility assay in which one monitors the movement of myosin-coated beads on an actin substratum which is present in Nitella cells which have been microdissected to remove the cell walls.